Nucleic acid arrays have become an increasingly important tool in the biotechnology industry and related fields. Nucleic acid arrays, in which a plurality of nucleic acids are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. One important use of nucleic acid arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
In methods of differential gene expression, arrays find use by serving as a substrate to which is bound nucleic acid “probe” fragments. One then obtains “targets” from at least two different cellular sources which are to be compared, e.g. analogous cells, tissues or organs of a healthy and diseased organism. The targets are then hybridized to the immobilized set of nucleic acid “probe” fragments. Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources.
A number of different physical parameters of the array which is used in such assays can have a significant effect on the results that are obtained from the assay. One physical parameter of nucleic acid arrays that can exert a significant influence over the nature of the results which are obtained from the array is probe size, i.e. the length of the individual probe nucleic acids stably associated with the surface of the solid support in the array. There are generally two different types of arrays currently finding use—(1) cDNA arrays, in which either full length or partial cDNAs are employed as probes; and (2) oligonucleotide arrays, in which probes of from about 8 to 25 nucleotides are employed.
In currently used cDNA arrays, the double stranded cDNAs which may be substantially full length or partial fragments thereof are stably associated with the surface of a solid support, e.g. nylon membrane. Advantages of cDNA arrays include high sensitivity, which features stems from the high efficiency of binding of the cDNA probe to its target and the stringent hybridization and washing conditions that may be employed with such arrays. Disadvantages of cDNA arrays include difficulties in large scale production of such arrays, low reproducibility of such arrays, and the like.
The other current alternative, oligonucleotide arrays, employs oligonucleotide probes in which each probe ranges from about 8 to 25, usually 20 to 35 nucleotides in length. While such arrays are more amenable to large scale production, they suffer from disadvantages as well. One significant disadvantage for such arrays is their lower sensitivity for target nucleic acids, as compared to cDNA arrays. Another disadvantage is the wide variation in hybridization efficiency of different probes for the same target in a given protocol, which feature requires the use of multiple oligonucleotide probes for the same target, which redundancy adds significantly to the cost of producing such arrays.
As such, there is a continued interest in the development of new array formats. Of particular interest would be the development of array format which combined the high sensitivity of cDNA arrays with the high throughput manufacturability of oligonucleotide arrays, where the format would not suffer from the disadvantages experienced with cDNA and oligonucleotide arrays, as described above.
Relevant Literature
Patents and patent applications of interest include: U.S. Pat. Nos. 5,143,854; 5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613; 5,470,710; 5,472,672; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,527,681; 5,529,756; 5,545,531; 5,547,839; 5,554,501; 5,556,752; 5,561,071; 5,580,726; 5,580,732; 5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134; 5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940; 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928; 5,847,219; 5,876,932; 5,919,626; WO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO 96/31622; WO 97/10365; WO 97/27317; WO 99/09217 WO 99/35505; EP 373 203; EP 742 287; EP 785 280; EP 799 897 and UK 8 803 000. References of interest include: Southern, et al. Nature Genet. (1999) 21:5–9; Sohail, et al., RNA (1999) 5:646-655; Mir et al., Nature Biotech. (1999)17: 788–792; Beier, et al., Nucl. Acids Res. (1999) 27:1970–1977; Rogers, et al., Anal. Biochem. (1999) 266:23–30; Vasiliskov, et al. BioTechniques (1999) 27:592–606; Chen, et al., Maldonado-Rodriguez, et al., Molec. Biotech.(1999) 11:13–25; Lipshutz, et al., Nature Genet. 1999, 21:20–24; Alon, et al., Proc. Natl. Acad. Sci. (1999) 96:6745–6750; Gunderson, et. al., Genome Research (1998) 8:1142–1153; Gilles et al., Nature Biotech. (1999) 17:365–370; Duggan, et al., Nature Genet. (1999) 21:10–14, Brown, P. O., Nature Genet (1999) 21:33–37; Pollack, et al., Nature Genet. (1999) 23:41–46; Wang et al., Gene (1999) 229:101–108; Bowtell, Nature Genet. (1999) 21:25–32; Schena, et al., TIBS (1998) 16:301–306; Debouck et al., Nature Genet. (1999) 21:48–50; The Microarray Meeting. Technology, Application and Analysis. Mountain Shadows Marriott Resort Scottsdale, Ariz., Sep. 22–25, 1999. Abstracts: 6–85; Gerhold et al., Trends Graves et al., Trends in Biotech. (1999) 17:127–134; Ekins et al., Trends in Biotech. (1999,) 17:217–218; Atlas Human cDNA Expression Array I (Apr. 1997) CLONTECHniques XII: 4–7; Lockhart et al., Nature Biotechnology (1996) 14: 1675–1680; Shena et al., Science (1995) 270:467–470; Schena et al., Proc. Nat'l Acad. Sci. U.S.A. (1996)93:10614–10619; and Chalifour et al., Anal. Biochem. (1994) 216:299–304.